Structural comparison of tRNA m1A58 methyltransferases revealed different molecular strategies to maintain their oligomeric architecture under extreme conditions

Background

tRNA m¹A58 methyltransferases (TrmI) catalyze the transfer of a methyl group from S-adenosyl-L-methionine to nitrogen 1 of adenine 58 in the T-loop of tRNAs from all three domains of life. The m¹A58 modification has been shown to be essential for cell growth in yeast and for adaptation to high temperatures in thermophilic organisms. These enzymes were shown to be active as tetramers. The crystal structures of five TrmIs from hyperthermophilic archaea and thermophilic or mesophilic bacteria have previously been determined, the optimal growth temperature of these organisms ranging from 37°C to 100°C. All TrmIs are assembled as tetramers formed by dimers of tightly assembled dimers.

Results

In this study, we present a comparative structural analysis of these TrmIs, which highlights factors that allow them to function over a large range of temperature. The monomers of the five enzymes are structurally highly similar, but the inter-monomer contacts differ strongly. Our analysis shows that bacterial enzymes from thermophilic organisms display additional intermolecular ionic interactions across the dimer interfaces, whereas hyperthermophilic enzymes present additional hydrophobic contacts. Moreover, as an alternative to two bidentate ionic interactions that stabilize the tetrameric interface in all other TrmI proteins, the tetramer of the archaeal P. abyssi enzyme is strengthened by four intersubunit disulfide bridges.

Conclusions

The availability of crystal structures of TrmIs from mesophilic, thermophilic or hyperthermophilic organisms allows a detailed analysis of the architecture of this protein family. Our structural comparisons provide insight into the different molecular strategies used to achieve the tetrameric organization in order to maintain the enzyme activity under extreme conditions.     

Hyperthermophiles and the problem of DNA instability

Rates of chemical decomposition of DNA at the optimal growth temperatures of hyperthermophiles seem incongruent with the requirements of accurate genome replication. The peculiar physiology, ecology and phylogeny of hyperthermophiles combine to suggest that these prokaryotes have solved a molecular problem (spontaneous loss of native DNA structure) of a magnitude that well-studied microorganisms do not face. The failure of DNA base composition to correlate with optimal growth temperature among hyperthermophiles provides indirect evidence that other mechanisms maintain their chromosomal DNA in the duplex form. Studies in vitro indicate that DNA primary structure is more difficult to maintain at extremely high temperature than is secondary structure, yet hyperthermophiles exhibit only modest levels of spontaneous mutation. Radiation sensitivity studies also indicate that hyperthermophiles repair their DNA efficiently in vivo , and underlying mechanisms are beginning to be examined. Several enzymes of DNA metabolism from hyperthermophilic archaea exhibit unusual biochemical features that may ultimately prove relevant to DNA repair. However, genomic sequencing results suggest that many DNA repair genes of hyperthermophilic archaea may not be recognized because they are not sufficiently related to those of well-studied organisms.     

Acetyl-CoA synthetase (ADP forming) in archaea,a novel enzyme involved in acetate formation and ATP synthesis

The anaerobic hyperthermophilic archaea Desulfurococcus amylolyticus, Hyperthermus butylicus, Thermococcus celer, Pyrococcus woesei, the hyperthermophilic bacteria Thermotoga maritima and Clostridium thermohydrosulfuricum and the aerobic mesophilic archaeon Halobacterium saccharovorum were grown either on complex media, on sugars or on pyruvate as carbon and energy sources. During growth acetate was formed as fermentation product by all organisms. The enzymes involved in acetyl-CoA formation from pyruvate and in acetate formation from acetyl-CoA were investigated:

1. Cell extracts of all species, both archaea and bacteria, catalyzed the coenzyme A-dependent oxidative decarboxylation of pyruvate with viologen dyes or with Clostridium pasteurianum ferredoxin as electron acceptors indicating a pyruvate: ferredoxin oxidoreductase to be operative in acetyl-CoA formation from pyruvate.
2. Cell extracts of all archaeal species, both hyperthermophiles (D. amylolyticus, H. butylicus, T. celer, P. woesei) and the mesophile H. saccharovorum, contained an acetyl-CoA synthetase (ADP forming), which catalyzes both acetate formation from acetyl-CoA and ATP synthesis from ADP and phosphate (P_i): Acetyl-CoA+ADP+P_i?Acetate + ATP+CoA. Phosphate acetyltransferase and acetate kinase could not be detected.
3. Cell extracts of the hyperthermophilic (eu)bacteria T. maritima and C. thermohydrosulfuricum contained phosphate acetyltransferase and acetate kinase rather than acetyl-CoA synthetase (ADP forming).

These data indicate that acetyl-CoA synthetase (ADP forming) represents a typical archaeal property rather than an enzyme specific for hyperthermophiles. It is proposed that in all acetate forming archaea the formation of acetate and of ATP from acetyl-CoA, ADP and P_i are catalyzed by acetyl-CoA synthetase (ADP forming), whereas in all acetate forming (eu)bacteria these reactions are catalyzed by two enzymes, phosphate acetyltransferase and acetate kinase.     

Potential to exploit postmortem enzyme degradation for evaluating arthropod viability

Inspecting for live organisms is the main method used to verify efficacy of phytosanitary treatments. Evaluating whether small, immobile organisms such as eggs, pupae and scale insects are alive or dead usually involves either checking morphological criteria or rearing them to observe development. These methods can be inaccurate, impractical and time consuming; thus, better methods are needed. To evaluate the potential for developing enzyme-based viability assays, we used electrophoretic gels to evaluate postmortem degradation of ten enzymes in Musca domestica L. (Diptera: Muscidae), four in Bemisia flocculosa Gill and Holder (Hemiptera: Aleyrodidae), and seven in Listronotus bonariensis (Kuschel) (Coleoptera: Curculionidae). Fresh insects displayed strong enzyme activity and distinct bands, but dead insects exhibited either no activity or weakened activity with reduced band resolution and increased migration of stained areas. Of ten enzymes investigated, seven showed clear indications of degradation just 1 day postmortem. Polyacrylamide gel electrophoresis of enzymes can be used to evaluate organism viability and has potential for estimating postmortem intervals. We also measured postmortem degradation rates of five M. domestica enzymes by assaying them in solution; these showed constant or gradually declining activity for 28 days postmortem, so live and dead specimens were less easily distinguished. By assaying enzymes in solution, it is possible to develop quick, easily operated tests that can be used outside the laboratory for a variety of quarantine-related purposes.     

The DNA content of sperm and hemocyte nuclei of the silkworm,Bombyx mori L.

To estimate the size of the haploid genome of the silkworm, Bombyx mori (Lepidoptera), amounts of Feulgen-DNA staining in individual nuclei of primary spermatocytes, spermatids, maturing sperm, and larval or pupal hemocytes were determined with an integrating microdensitometer and compared with the Feulgen-DNA levels found for chicken erythrocyte nuclei, or the sperm and erythrocyte nuclei of Xenopus laevis that were included with each Bombyx preparation as empirical reference standards of 2.5, 3.15, and 6.3×10^?12 g DNA per cell, respectively. Under these conditions, the haploid male genome of B. mori was estimated as 0.52±0.01 (S.E.)×10^?12 g DNA, corresponding to a molecular weight of roughly 3.1×10¹¹ daltons. From similar measurements of Feulgen-stained hemocyte nuclei, approximately 1.0±0.05 (S.E.)×10^?12 g DNA was estimated for the diploid or 2C male genome of Bombyx. These values compare favorably with estimates of genome size based upon analysis of the kinetics of reassociation of DNA isolated from B. mori and provide an independent basis for assessing the degree of polyploidy achieved by the giant nuclei in the posterior silk gland prior to its secretion of fibroin at the end of the fifth larval instar.     

The Bombyx mori genome: analysis by DNA reassociation kinetics

The size and nucleotide sequence complexity of the Bombyx mori genome has been determined from the kinetics of reassociation of its DNA. Nonrepeated DNA comprises 55% of the genome, and the remainder is divided equally between sequences repeated roughly 500 and 50000 times. Non-repeated sequence DNA virtually free of repeated sequences was prepared by partial reassociation and subsequent fractionation on hydroxyapatite. The nucleotide sequence complexity of this component was determined relative to DNA from B. subtilis and E. coli. After correction for the size of the repeated sequence fraction, the DNA content of the Bombyx mori genome was calculated to be 0.53±0.02×10^?12 g. This value compares favorably with the DNA content of haploid B. mori spermatids and mature sperm determined cytophotometrically by Rasch (1973).     

Protein-Protein Interactions Leading to Recruitment of the Host DNA Sliding Clamp by the Hyperthermophilic Sulfolobus islandicus Rod-Shaped Virus 2

Viruses infecting hyperthermophilic archaea typically do not encode DNA polymerases, raising questions regarding their genome replication. Here, using a yeast two-hybrid approach, we have assessed interactions between proteins of Sulfolobus islandicus rod-shaped virus 2 (SIRV2) and the host-encoded proliferating cell nuclear antigen (PCNA), a key DNA replication protein in archaea. Five SIRV2 proteins were found to interact with PCNA, providing insights into the recruitment of host replisome for viral DNA replication.     

Metabolic reconstruction and experimental verification of glucose utilization in <Emphasis Type="Italic">Desulfurococcus amylolyticus</Emphasis> DSM 16532

Desulfurococcus amylolyticus DSM 16532 is an anaerobic and hyperthermophilic crenarchaeon known to grow on a variety of different carbon sources, including monosaccharides and polysaccharides. Furthermore, D. amylolyticus is one of the few archaea that are known to be able to grow on cellulose. Here, we present the metabolic reconstruction of D. amylolyticus’ central carbon metabolism. Based on the published genome, the metabolic reconstruction was completed by integrating complementary information available from the KEGG, BRENDA, UniProt, NCBI, and PFAM databases, as well as from available literature. The genomic analysis of D. amylolyticus revealed genes for both the classical and the archaeal version of the Embden-Meyerhof pathway. The metabolic reconstruction highlighted gaps in carbon dioxide-fixation pathways. No complete carbon dioxide-fixation pathway such as the reductive citrate cycle or the dicarboxylate-4-hydroxybutyrate cycle could be identified. However, the metabolic reconstruction indicated that D. amylolyticus harbors all genes necessary for glucose metabolization. Closed batch experimental verification of glucose utilization by D. amylolyticus was performed in chemically defined medium. The findings from in silico analyses and from growth experiments are discussed with respect to physiological features of hyperthermophilic organisms.     

Role of PARP2 in DNA repair

The genome stability of higher eukaryotes depends largely on the functioning of the DNA repair systems. In turn, the precise regulation of each step of repair processes is necessary for the efficient DNA repair. Although most pathways of DNA repair have already been established, their regulation mechanisms require further investigation. Poly(ADP-ribose) polymerases (PARPs) are widely considered to be potential regulators of DNA repair. The role of the most prominent member of this protein family, i.e., PARP1, in DNA repair has been being intensively studied, while the literature data on participation in the repair processes of PARP2, the closest PARP1 homolog, are poorly summarized, although a great body of information concerning its participation in DNA repair has been accumulated. Using the PARP2-deficient model organisms and cell lines, their increased sensitivity to several DNA damaging agents was elucidated. The accumulation of PARP2 at the DNA damage sites in cells was shown. There are data that demonstrate the proteinprotein interaction of PARP2 with several proteins of the base excision repair/single-strand break repair and nonhomologous end joining. Most of the data on the PARP2 role were obtained in experiments with model organisms and cell lines; thus, it is difficult to elucidate the influence of PARP2 on specific processes in vivo. In this review, we tried to summarize data on the participation of PARP2 in the DNA repair processes, including our recent results.     

The Reverse Gyrase from Pyrobaculum calidifontis,a Novel Extremely Thermophilic DNA Topoisomerase Endowed with DNA Unwinding and Annealing Activities

Reverse gyrase is a DNA topoisomerase specific for hyperthermophilic bacteria and archaea. It catalyzes the peculiar ATP-dependent DNA-positive supercoiling reaction and might be involved in the physiological adaptation to high growth temperature. Reverse gyrase comprises an N-terminal ATPase and a C-terminal topoisomerase domain, which cooperate in enzyme activity, but details of its mechanism of action are still not clear. We present here a functional characterization of PcalRG, a novel reverse gyrase from the archaeon Pyrobaculum calidifontis. PcalRG is the most robust and processive reverse gyrase known to date; it is active over a wide range of conditions, including temperature, ionic strength, and ATP concentration. Moreover, it holds a strong ATP-inhibited DNA cleavage activity. Most important, PcalRG is able to induce ATP-dependent unwinding of synthetic Holliday junctions and ATP-stimulated annealing of unconstrained single-stranded oligonucleotides. Combined DNA unwinding and annealing activities are typical of certain helicases, but until now were shown for no other reverse gyrase. Our results suggest for the first time that a reverse gyrase shares not only structural but also functional features with evolutionary conserved helicase-topoisomerase complexes involved in genome stability.     

Comparative electrophoretical analysis of plasmid-like mitochondrial DNAs in Vicia faba and in some other legumes

Minicircular DNAs found in mitochondria of 6-d-old etiolated seedlings of Vicia faba L. were also present in mitochondria isolated from cell suspension cultures and from green leaves of this plant. These results support the suggestion that the plasmid-like molecules found in mitochondria of V. faba are an essential component of the mitochondrial genome. The minicircular DNAs were, apparently, peculiar for the species V. faba since they were found in all three cultivars of this species which were studied. The distribution pattern of plasmid-like DNAs in Vicia villosa L. was completely different and mitochondria from Medicago sativa L. also contained specific minicircular DNAs. Thus, minicircular DNAs are typical for the mitochondrial genomes of several legumes and different plant species have their specific mitochondrial plasmid-like DNAs     

Chromosomal localization and genomic organization of cloned repetitive DNA fragments in mosquitoes (Diptera: Culicidae)

The chromosomal localization and genomic organization of three cloned repetitive DNA fragments (viz., H-76, H-61, and H-19) isolated from theAedes albopictus genome have been examined inAe. albopictus and six otherAedes species:Ae. aegypti, Ae. seatoi, Ae. flavopictus, Ae. polynesiensis, Ae. alcasidi andAe. katherinensis. The results fromin situ and Southern hybridization analyses show that the sequences homologous to cloned repetitive DNA fragments are dispersed throughout the genome in each species. The sequences homologous to these cloned repetitive DNA fragments are also found inHaemagogus equinus, Tripteroides bambusa andAnopheles quadrimaculatus and are dispersed in their genomes. Data indicate divergence in the amount and the structural organization of sequences homologous to these cloned fragments among mosquito species.     

Nonspecificity of some commonly used inhibitors of DNA synthesis in cultures ofStreptococcus faecalis

The specificity of three commonly used inhibitors of DNA synthesis were tested in the batch culture ofStreptococcus faecalis ATCC 8043 in rich broth medium. It was shown that nalidixic acid, mitomycin C and 6-(4-hydroxyphenylazo)uracil inhibit the cell mass as much as they decrease net DNA synthesis. Hence the drugs tested are highly unspecific inhibitors of DNA synthesis inS. faecalis; i.e. they all interfere with other processes as well as with DNA synthesis.     

Genome DNA content and chromosome organization in Gossypium

The ascending genome size in Gossypium is assumed to be D, A, B, E and F, and C. Feulgen cytophotometry revealed that mean value of DNA content for each genome was D= 10.95, B = 13.88, F = 14.31, E = 18.24, A = 18.66, and C = 20.30, and that there is a close relationship of genomic chromosome size and DNA content. Evidence suggests that the five genomes with large chromosomes arose from a D genome-like progenetor by large scale, saltatory replication of repetitive DNA distributed uniformly through the ancestral genome. Corresponding adjustment in recombination units did not accompany the two-fold divergence in DNA value of the two homoeologous A and D genomes in the allotetraploid species.     

Enhanced acetate ester production of Chinese liquor yeast by overexpressing ATF1 through precise and seamless insertion of PGK1 promoter

As the most important group in the flavor profiles of Chinese liquor, ester aroma chemicals are responsible for the highly desired fruity odors. Alcohol acetyltransferase (AATase), which is mainly encoded by ATF1, is one of the most important enzymes for acetate ester synthesis in Saccharomyces cerevisiae. In this study, we overexpressed ATF1 in Chinese liquor yeast through precise and seamless insertion of PGK1 promoter (PGK1p) via a novel fusion PCR-mediated strategy. After two-step integration, PGK1p was embedded in the 5′-terminal of ATF1 exactly without introduction of any extraneous DNA sequence. In the liquid fermentation of corn hydrolysate, both mRNA level and AATase activity of ATF1 in mutant were pronounced higher than the parental strain. Meanwhile, productivity of ethyl acetate increased from 25.04 to 78.76 mg/l. The self-cloning strain without any heterologous sequences residual in its genome would contribute to further commercialization of favorable organoleptic characteristics in Chinese liquor.     

An intermolecular disulfide bond is required for thermostability and thermoactivity of β-glycosidase from Thermococcus kodakarensis KOD1

Scientists are interested in understanding the molecular origin of protein thermostability and thermoactivity for possible biotechnological applications. The enzymes from extremophilic organisms have been of particular interest in the last two decades. β-glycosidase, Tkβgly is a hyperthermophilic enzyme from Thermococcus kodakarensis KOD1. Tkβgly contains two conserved cysteine residues, C88 and C376. The protein tertiary structure obtained through homology modeling suggests that the C88 residue is located on the surface whereas C376 is inside the protein. To study the role of these cysteine residues, we substituted C88 and C376 with serine residues through site-directed mutagenesis. The wild-type and C376S protein existed in dimeric form and C88S in monomeric form, in an SDS-PAGE gel under non-reducing conditions. Optimal temperature experiments revealed that the wild-type was active at 100 °C whereas the C88S mutant exhibited optimal activity at 70 °C. The half-life of the enzyme at 70 °C was drastically reduced from 266 h to less than 1 h. Although C88 was not present in the active site region, the k _cat/K _m of C88S was reduced by 2-fold. Based on the structural model and biochemical properties, we propose that C88 is crucial in maintaining the thermostability and thermoactivity of the Tkβgly enzyme.